The mycobacterial cell wall contains large amounts of unusual lipids, including mycolic acids that are covalently linked to the underlying arabinogalactanpeptidoglycan complex. Hydrocarbon chains of many of these lipids have been shown to be packed in a direction perpendicular to the plane of the cell surface. In this study, we examined the dynamic properties of the organized lipid domains in the cell wall isolated from Mycobacterium chelonae grown at 30x C. Differential scanning calorimetry showed that many of the lipids underwent major thermal transitions between 30x and 65x C, which is above the growth temperature; a result suggesting that a significant portion of the lipids existed in a structure of extremely low fluidity in growing cells. Spin-labeled fatty acid probes were successfully inserted into the more fluid part of the cell wall. Our model of the cell wall suggests that this domain corresponds to the outermost leaflet, a conclusion reinforced by the observation that labeling of the intact cells produced electron spin resonance spectra similar to those of the isolated cell wall. Use of stearate, labeled at different positions, showed that the fluidity within the outer leaflet increased only slightly as the nitroxide group was placed farther away from the surface. These results are consistent with the model of mycobacterial cell wall containing an asymmetric lipid bilayer, with an internal less fluid mycolic acid leaflet and an external more fluid leaflet composed of lipids containing shorter chain fatty acids. The presence of the low-fluidity layer will lower the permeability of the cell wall to lipophilic antibiotics and chemotherapeutic agents and may contribute to the well known intrinsic resistance of mycobacteria to such compounds.